Intracellular Ca2+ signals are an integral part of information processing by neuronal circuits facilitating pre- and postsynaptic functions including long-term potentiation (LTP) and depression (LTD) of synaptic transmission, the electrophysiological correlates of long-term synaptic plasticity. Since finely tuned changes in Ca2+ modulate a variety of intracellular functions, signal specificity requires a tight spatial and temporal control of the cytosolic Ca2+ signal. This need is most apparent for fast neurotransmitter release where voltage-dependent Ca2+ signaling triggers the fusion of synaptic vesicles at presynaptic terminals on a sub-millisecond scale. The endoplasmic reticulum (ER) can act as a Ca2+ store/sink or as a Ca2+ source and is likely to balance these dual roles at presynaptic terminals to meet the specific needs of the synapse for transmitter release. However, the contributions of ER to presynaptic Ca2+ signaling and neurotransmitter release are not well understood. We hypothesize that the ER acts as the primary presynaptic Ca2+ sink at fly NMJs. If so, the presynaptic ER may not only temporarily buffer cytosolic Ca2+ by SERCA pumping but also permanently remove Ca from synaptic terminals by "tunneling" luminal Ca2+ into axons. Limited ER Ca2+ release within terminals may modulate neurotransmitter release but mainly serve mitochondrial Ca2+ uptake activating mitochondrial energy production. We will test this hypothesis by exploiting genetically manipulated Drosophila and image depolarization-induced changes in presynaptic Ca levels of the ER lumen, mitochondria and the cytosol of larval NMJs. At the center of this proposal is the development of transgenic Ca2+ indicators that can faithfully report Ca2+ changes in the lumen of the ER and mitochondria, which we expect will also be of wide use for the genetic analysis of mitochondrial and/or ER Ca2+ dynamics in any cell of Drosophila. Specifically, we will determine the dynamics of ER-mediated Ca2+ uptake, Ca2+ release and/or Ca2+ diffusion (tunneling) upon repetitive nerve stimulation at presynaptic terminals of larval Drosophila NMJs (Aim 1) and determine the role of ER-mitochondria interactions for presynaptic Ca2+ homeostasis at larval Drosophila NMJs (Aim 2). From this systematic analysis a basic framework will emerge for better understanding the role of the ER in presynaptic Ca2+ signaling/homeostasis at presynaptic terminals expanding our understanding of important regulatory mechanisms of synaptic transmission and their relation to the functional plasticity of the nervous system and human health. [unreadable] [unreadable]